Carrot variety purple royale

ABSTRACT

The invention relates to the carrot variety designated Purple Royale. Provided by the invention are the seeds, plants, and derivatives of the carrot variety Purple Royale. Also provided by the invention are tissue cultures of the carrot variety Purple Royale and the plants regenerated therefrom. Still further provided by the invention are methods for producing carrot plants by crossing the carrot variety Purple Royale with itself or another carrot variety and plants produced by such methods.

This application claims the benefit of priority of United StatesProvisional Application No. 62/794,230, filed Jan. 18, 2019, thedisclosure of which is hereby incorporated by reference as if writtenherein in its entirety.

FIELD OF THE INVENTION

The present invention relates to the field of plant breeding. Inparticular, the invention provides carrot plants with a high anthocyaninlevel in the roots. The invention further provides for a new anddistinct carrot variety designated Purple Royale and for breedingmethods with these plants.

BACKGROUND OF THE INVENTION

Carrot (Daucus carota subsp. sativus), is a root vegetable, usuallyorange in color, though purple, red, white, and yellow varieties exist.It has a crisp texture when fresh. The most commonly eaten part of acarrot is a taproot, although the greens are edible as well. It is adomesticated form of the wild carrot Daucus carota, wildly believed tobe from central Asia. Most of the modern breeding of the domestic carrotoriginated in Europe and which has focused on the selection of a greatlyenlarged and more palatable, less woody-textured edible taproot. Theannual purple varieties essentially missed out on those centuries ofmodern breeding until approximately the last 20 years. The worldproduction of carrots and turnips for calendar year 2011 was almost35.658 million tons (Food and Agriculture Organization of the UnitedNations (FAO)).

The aim of a vegetable breeder is to combine desirable traits in asingle variety. Such desirable traits may include any trait deemedbeneficial by a grower and/or consumer, including greater yield,resistance to insects or disease, tolerance to environmental stress, andnutritional value.

Total anthocyanin level is an example of such a desirable trait.Anthocyanins are polyphenols with known antioxidant activity which maybe responsible for some biological activities including the preventionor lowering the risk of cardiovascular disease, diabetes, arthritis andcancer. Nevertheless, such properties, their stability andbioavailability depend on their chemical structure (Miguel MG. Journalof Applied Pharmaceutical Science. 01 (06); 2011: 7-15.

A uniform population of a breeding line can be obtained byself-pollination and selection for type. Plants thus obtained becomehomozygous at almost all gene loci, i.e. a homozygous plant. Crossingtwo such plants of different genotypes produces a uniform population ofplants that are heterozygous for many loci. On the other hand, a crossof two plants each heterozygous at a number of loci produces apopulation of plants that differ genetically and are not uniform. Due tothis non-uniformity, performance of such plants is unpredictable.

Thus, a vegetable breeder prefers development of a homozygous inbredplant that can be crossed to produce uniform varieties. Pedigreebreeding and recurrent selection are examples of breeding methods thathave been used to develop inbred plants from breeding populations. Thosebreeding methods combine the genetic backgrounds from two or more plantsor various other broad-based sources into breeding pools from which newlines derived therefrom are developed by selfing and selection ofdesired phenotypes. The new lines are evaluated to determine which ofthose have commercial potential.

So far, breeding efforts have provided a number of useful carrot lineswith beneficial traits, however, there remains a great need in the artfor new lines with further improved traits. There is thus a need for newcarrot varieties having specific combination of trait or color.

SUMMARY OF THE INVENTION

The objective of the invention was to develop an Imperator type varietywith purple coloration throughout and a high anthocyanin content of themature root.

In one aspect, the present invention provides seed of a new carrot(Daucus carota) variety, designated Purple Royale, having been depositedunder Accession Number ______, a plant, or a part thereof, produced bygrowing said seed. The invention also provides methods and compositionsrelating to plants, plant parts, seeds and progenies of carrot varietyPurple Royale.

Variety Purple Royale is most similar to comparison variety Anthonina orDeep Purple, which is a variety produced and sold by Bejo. However,Purple Royale differs from Anthonina, Purple Elite, Purple Haze, andDeep Purple in one or more, e.g., at least two, at least three,optionally all morphological and/or physiological characteristics listedin the following (see also Table 1), when grown under the sameenvironmental conditions:

an (average) root core thickness (at midpoint of cross-section) that isat least about 12%, or preferably at least about 13%, 14%, 15%, 16%,17%, 18%, or even about 19% bigger than the core thickness of one ormore of black carrot varieties Anthonina, Purple Elite, Purple Haze, andDeep Purple;

a (average) root diameter at midpoint that is at least about 7.5%, orpreferably at least about 10%, 11%, 12%, 13%, 14%, or even about 14.9%smaller than the diameter at midpoint of one or more of black carrotvarieties Anthonina, Purple Elite, Purple Haze, and Deep Purple;

a (average) carrot root length (minus taproot) that is at least about5%, or preferably at least about 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, or evenabout 8.3% smaller than the carrot length of one or more of black carrotvarieties Anthonina, Purple Elite, Purple Haze, and Deep Purple;

a purple cross-section interior color of both core and cortex, which isdarker than the purple cross-section interior color of Anthonina, PurpleElite, Purple Haze, and Deep Purple, e.g. N79A for Purple Royale versusthe designation listed for the other carrot varieties in Table 1;

Other differences between variety Purple Royale and Anthonina, PurpleElite, Purple Haze, and Deep Purple reveals that Purple Royale alsodiffers significantly from Anthonina, Purple Elite, Purple Haze, andDeep Purple in one or more, e.g., at least two, at least three,optionally all morphological and/or physiological characteristics listedin the following (see also Table 1), when grown under the sameenvironmental conditions.

The present invention provides a carrot plant variety designated,representative seed of said variety having been deposited under ATCCAccession Number ______, and plant parts of the new variety such as forexample seed, leaf, pollen, an ovule, taproot, root and a cell of theplant.

The invention also concerns the seed of the carrot Purple Royale,representative seed of said variety having been deposited under ATCCAccession Number ______, a plant, or a part thereof, produced by growingsaid seed. Or carrot plants having all or essentially all thephysiological and morphological characteristics of Purple Royale.

The invention further relates to breeding methods using plants or seedof carrot variety Purple Royale.

Additional objects, features and advantages of the present inventionwill become apparent from the following detailed description. It shouldbe understood, however, that the detailed description and any specificexamples provided, while indicating specific embodiments of theinvention, are given by way of illustration only, since various changesand modifications within the spirit and scope of the invention willbecome apparent to those skilled in the art from this detaileddescription.

DETAILED DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 shows the presence of anthocyanin in Purple Royale.

FIG. 2 shows the presence/absence of anthocyanin in Purple Royale,Anthonina, Purple Elite, Purple Haze, and Deep Purple

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a carrot plant, or part thereof, theroots of said plant. The invention further relates to plant parts ofthese plants such as for example seed, leaf, pollen, an ovule, taproot,root and a cell of the plant. In another aspect, the plant parts includeleaf, pollen, an ovule, taproot, root and a cell of the plant. In yetanother aspect, the plant part is a root.

The invention also includes seed of the plant of the plants of theinvention, i.e seeds from which a carrot plant, the roots of whichcomprise, can be grown.

In another embodiment the invention concerns a plant according to theinvention wherein the genetic elements conferring said total anthocyaninlevel is obtainable from carrot variety Purple Royale, a representativesample of seed of Purple Royale having been deposited under ATCCAccession Number ______.

In still another embodiment the invention relates to a plant accordingto the invention wherein the genetic elements conferring said areobtainable from carrot variety Purple Royale, a representative sample ofseed of Purple Royale having been deposited under ATCC Accession Number______.

Anthocyanin content is a function of growth stage and productionenvironment.

In another aspect the root of the carrot plant of the invention has atotal average anthocyanin level of at least about 20 ppm or preferablyat least about 21 ppm, 22 ppm, 23 ppm, 24 ppm, 25 ppm, or even about 26ppm. The anthocyanin content of the carrot variety Purple Royale willexceed any known carrot variety at all growth stages.

All parts of the specification which refer herein to variety PurpleRoyale, such as breeding, progeny, plant parts, cells, seeds,development of identification of EDVs, etc. can also be applied in amore general way, i.e. to methods for transfer the genetic elementsconferring high anthocyanin levels. Thus, the invention not only relatesto the above carrot plant, but also to cells, tissues, plant parts,seeds, EDVs, progeny, transformed carrot plants, etc. which comprise thegenetic determinants obtainable from (obtained from; as present in)Purple Royale which confer the high anthocyanin content and optionallythe genetic elements which confer the faint haloing and zoning.

The present invention provides a carrot plant variety designated PurpleRoyale, representative seed of said variety having been deposited underATCC Accession Number and plant parts of the new variety such as forexample seed, leaf, pollen, an ovule, taproot, root and a cell of theplant. In another aspect, the plant parts include leaf, pollen, anovule, taproot, root and a cell of the plant. In yet another aspect, theplant part is a root. Also provided are carrot plants having all oressentially all the physiological and morphological characteristics ofsuch a plant, i.e. a carrot plant that does not differ (statistically)significantly from Purple Royale in the morphological and/orphysiological characteristics of Table 1, when grown under the sameconditions.

The invention also concerns the seed of the carrot Purple Royale,representative seed of said variety having been deposited underAccession Number ATCC ______, a plant, or a part thereof (e.g. a root),produced by growing said seed. Or carrot plants having all oressentially all the physiological and/or morphological characteristicsof Purple Royale when grown under the same conditions. The carrot seedof the invention (i.e. seed from which a plant of variety Purple Royalecan be grown) may be provided as an essentially homogeneous populationof carrot seed according to the invention. Essentially homogeneouspopulations of seed are generally free from substantial numbers of otherseed. Therefore, seed of the carrot Purple Royale or carrot plantshaving all or essentially all the physiological and/or morphologicalcharacteristics of Purple Royale may be defined as forming at leastabout 97% of the total seed, including at least about 98%, 99% or moreof the seed. The seed population may be separately grown to provide anessentially homogeneous population of carrot plants according to theinvention. Also provided are plants or plant parts such as seed(produced on the plant Purple Royale, e.g. after self-pollination orcross-pollination by another carrot plant), leaf, pollen, an ovule,taproot, roots or a cell produced by growing the seeds of the invention.In yet another aspect, the plant part is a root.

In another aspect the invention relates to a variety of Purple Royalehaving at least one, two or three physiological and/or morphologicalcharacteristics which are (statistically significantly) different fromthose of Purple Royale and which otherwise has essentially allphysiological and morphological characteristics of a carrot plantdesignated Purple Royale, a representative sample of seeds of whichhaving been deposited under ATCC Accession Number ______.

In another aspect, the invention relates to a variety of Purple Royalehaving at least one or two physiological and/or morphologicalcharacteristics which are significantly different from those of PurpleRoyale and which otherwise comprises at least 3, 4 or 5 or more (or all)of the distinguishing characteristics 1)-5) as defined below, orpreferably at least 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 or all 12 of thedistinguishing characteristics 1)-12) of Purple Royale and/or hasessentially all physiological and morphological characteristics of acarrot plant designated Purple Royale obtainable by selecting a naturalor induced mutant, or a somaclonal variant, or a natural variant from apopulation of plants designated Purple Royale.

In still another aspect, a carrot plant, or a part thereof, is providedwhich does not significantly differ in distinguishing characteristics1)-5) of carrot plant Purple Royale when grown under the sameconditions. In yet another aspect, the invention relates to a carrotplant, or a part thereof, which does not significantly differ indistinguishing characteristics 1)-5) and additionally comprises (i.e.does not differ statistically significantly in) at least one, two, threeor all of distinguishing characteristics 6)-12) of carrot plant PurpleRoyale when grown under the same conditions.

In yet another aspect, the invention relates to a carrot plant, or apart thereof, which does not differ significantly from carrot plantPurple Royale in any of the physiological and/or morphologicalcharacteristics of Table 1 when grown under the same conditions.

In another aspect the invention relates to a carrot plant, or a partthereof, which does not significantly differ from carrot plant PurpleRoyale in any of the distinguishing characteristics (see USDAdescriptors) consisting of 1) average root core thickness at midpoint ofcross-section; 2) average root diameter at midpoint; 3) average carrotlength (minus taproot); 4) cross-section interior color of both core andcortex; 5) haloing and zoning of the root at market maturity, when grownunder the same conditions.

In still another aspect, a carrot plant is provided, which statisticallysignificantly differs from Purple Royale in at least one morphologicaland/or physiological characteristic, but which does not differsignificantly from Purple Royale in the following characteristics (seeUSDA descriptor) when grown under the same conditions: 1) average rootcore thickness at midpoint of cross-section; 2) average root diameter atmidpoint; 3) average carrot length (minus taproot); 4) cross-sectioninterior color of both core and cortex; 5) haloing and zoning of theroot at market maturity.)

In a further aspect, a carrot plant is provided, which statisticallysignificantly differs from Purple Royale in at least one morphologicaland/or physiological characteristics, but which does not differsignificantly from Purple Royale in the following characteristics (seeUSDA descriptors) when grown under the same conditions: 1) average rootcore thickness at midpoint of cross-section; 2) average root diameter atmidpoint; 3) average carrot length (minus taproot); 4) cross-sectioninterior color of both core and cortex; 5) haloing and zoning of theroot at market maturity; and which further does not significantly differfrom the plant designated Purple Royale in one, two, three or more ofthe following characteristics when grown under the same conditions: 6)Petiole length from crown to first pinna; 7) average length of taproot;8) leaf blade color (at harvest stage); 9) leaf blade divisions (atharvest stage); 10) leaf petiole anthocyanin level at harvest stage andpetiole pubescence at harvest stage; 11) average root diameter atshoulder; 12) total anthocyanin level.

In a further embodiment a carrot plant is provided, which (statisticallysignificantly) differs from the carrot plant designated Purple Royale,representative seeds of said carrot plant having been deposited underATCC accession number ______, in at least one, two, three, four, or fivemorphological and/or physiological characteristics when grown under thesame environmental conditions, whereby the morphological and/orphysiological characteristics are those of Table 1. The carrot plantdoes, thus, not differ in a statistically significant way from PurpleRoyale in any of the morphological and/or physiological characteristicsof Table 1 when grown under the same conditions, or only differs is one,two, three, four or five of the morphological and/or physiologicalcharacteristics of Table 1, while there is no significant difference inthe other characteristics.

In one embodiment a carrot plant is provided, designated Purple Royale,which does not (statistically significantly) differ in any of themorphological and/or physiological characteristics of Table 1 fromplants grown from seeds deposited under ATCC accession number ______when grown under the same environmental conditions.

In yet another aspect of the invention, a tissue culture or cell-cultureof regenerable cells of a carrot plant according to the invention isprovided. The tissue culture or cell-culture will preferably be capableof regenerating carrot plants capable of expressing all of thephysiological and/or morphological characteristics of the startingplant, and of regenerating plants having substantially the same genotypeas the starting plant. Examples of some of the physiological and/ormorphological characteristics of the carrot Purple Royale include thosetraits set forth in Table 1 herein when grown under the environmentalconditions outlined herein with reference to the data of Table 1. Theregenerable cells in such tissue or cell cultures may be derived, forexample, from carrot explants, such as embryos, meristems, petioles,cuttings, protoplasts, cotyledons, pollen, leaves, nodes, anthers,roots, taproots, root tips, pistils, flowers, seed and stems. Stillfurther, the present invention provides carrot plants regenerated from atissue culture or cell culture of the invention. These plants have allthe physiological and/or morphological characteristics of a plantaccording to the invention.

In another aspect, the above described carrot plants are obtainable fromin vitro cell or tissue cultures. As already elsewhere in thisapplication, in vitro cell or tissue cultures are known in the art andcan be used to either vegetatively reproduce the plant from which thecells or tissues were obtained or to identify and/or select a phenotypicvariant (such as an EDV), and to regenerate such a variant. Thephenotypic variant may, for example, be a somaclonal variant, mutant oroff-type, but is preferably genetically stable. Thus, the variantphenotype is preferably genetically stable, also in the mature plantsregenerated from the cell or tissue culture. That means, the phenotypicvariant does not show variation in phenotype which are transient and arenot genetically stable. Once selected, such selected variants can thenin turn also be reproduced true to type using in vitro cell or tissueculture or by propagation via seed.

Thus, in one aspect, a carrot plant is provided which is clonallypropagated (it is a vegetative reproduction) from Purple Royale cells ortissue and which comprises all the distinguishing characteristics ofPurple Royale when grown under the same environmental conditions. Inanother aspect it further comprises one or more of the furtherdistinguishing characteristics. In yet another aspect it comprises allmorphological and/or physiological characteristics of Purple Royale asgiven in Table 1. And in yet a further aspect it comprises allmorphological and/or physiological characteristics of Purple Royale asgiven in Table 1, except that it significantly differs from PurpleRoyale in one, two, three, four, or five of the morphological and/orphysiological characteristics of Table 1.

The invention also concerns methods of vegetatively propagating a plantof the invention. In certain embodiments, the method comprises the stepsof: (a) collecting tissue or cells capable of being propagated from aplant of the invention; (b) cultivating said tissue or cells to obtainproliferated shoots; and (c) rooting said proliferated shoots, to obtainrooted plantlets. Steps (b) and (c) may also be reversed, i.e. firstcultivating said tissue to obtain roots and then cultivating the tissueto obtain shoots, thereby obtaining rooted plantlets. The rootedplantlets may then be further grown, to obtain plants. In oneembodiment, the method further comprises step (d) growing plants fromsaid rooted plantlets.

In still yet another aspect of the invention, processes are provided forproducing carrot seeds, plants and roots, which processes generallycomprise crossing a first parent carrot plant with a second parentcarrot plant, wherein at least one of the first or second parent carrotplants is a plant according to the invention.

One embodiment of the invention refers to a method of producing a carrotplant comprising crossing a carrot plant of variety Purple Royale with asecond carrot plant one or more times such as one, two, three, four,five, six or more times. This method comprises in one embodimentselecting progeny from said crossing.

These processes may be further exemplified as processes for preparingthe carrot seed or plants, wherein a first carrot plant is crossed witha second carrot plant of a different, distinct genotype to provide aplant that has, as one of its parents, a plant of Purple Royale.

The present invention also provides the carrot seeds and plants producedby a process that comprises crossing a first parent carrot plant with asecond parent carrot plant, wherein at least one of the first or secondparent carrot plants is a plant provided herein, such as from thevariety Purple Royale. In another embodiment of the invention, carrotseed and plants produced by the process are first filial generation (F₁)carrot seed and plants produced by crossing a plant in accordance withthe invention with another, distinct plant. The present inventionfurther contemplates plant parts of such an F₁ carrot plant, and methodsof use thereof. Therefore, certain exemplary embodiments of theinvention provide an F₁ carrot plant and seed thereof.

In another embodiment of the invention, carrot variety Purple Royale iscrossed to produce seed of the variety designated Purple Royale. In anycross herein, either parent may be the male or female parent. In theseprocesses, crossing will result in the production of seed. The seedproduction occurs regardless of whether the seed is collected or not.

In certain embodiments, the invention provides methods of introducing adesired trait into a carrot plant comprising the steps of: (a) crossinga plant of variety Purple Royale with a second carrot plant thatcomprises a desired trait to produce F₁ progeny, (b) selecting an F₁progeny that comprises the desired trait(s), e.g., one, two, three ormore desired trait(s), (c) optionally selfing the F₁ progeny one or moretimes to produce F₂, F₃, or further generation selfing progeny, (d)crossing the selected F₁ progeny or the selfing progeny with a plant ofvariety Purple Royale to produce backcross progeny, and (e) selectingbackcross progeny comprising the desired trait(s) and which otherwisehas all or essentially all the physiological and morphologicalcharacteristics of carrot variety Purple Royale, (f) optionally, steps(d) and (e) can be repeated one or more times, e.g., three or more timessuch as three, four, five, six or seven times, in succession to producehigher backcross progeny (e.g., selected fourth, fifth, sixth, seventhor eighth or higher backcross progeny) that comprises the desired trait.The invention also provides carrot plants produced by these methods; arepresentative sample of seed of Purple Royale having been depositedunder ATCC Accession Number ______.

In another aspect of the invention, a carrot plant of variety PurpleRoyale comprising an added heritable trait (in addition to all themorphological and/or physiological characteristics of Purple Royale) ora modified heritable trait is provided, e.g., an Essentially DerivedVariety of Purple Royale (such as a mutant, or off-type, natural variantor somaclonal variant) having one, two or three physiological and/ormorphological characteristics which are different from those of PurpleRoyale and which otherwise has all the physiological and morphologicalcharacteristics of Purple Royale, wherein a representative sample ofseed of variety Purple Royale has been deposited under ATCC AccessionNumber ______. The heritable trait may comprise a genetic locus that is,for example, a dominant or recessive allele. In one embodiment of theinvention, a plant of the invention is defined as comprising a singlelocus conversion. For example, one, two, three or more heritable traitsmay be introgressed at any particular locus using a different allelethat confers the new trait or traits of interest. In specificembodiments of the invention, the single locus conversion confers one ormore traits such as, for example, herbicide tolerance, insectresistance, disease resistance and modulation of plant metabolism andmetabolite profiles. In further embodiments, the trait may be conferredby a naturally occurring gene introduced into the genome of the varietyby backcrossing, a natural or induced mutation, a somaclonal variant, anoff-type, or a transgene introduced through genetic transformationtechniques into the plant or a progenitor of any previous generationthereof. When introduced through transformation, a genetic locus maycomprise one or more genes integrated at a single chromosomal location.Thus, the invention comprises a method of producing a plant comprisingan added desired trait, the method comprising introducing a transgeneconferring a desired trait into a plant of carrot variety Purple Royale.

In still yet another aspect, the invention provides a method ofdetermining the genotype of a plant of the invention comprisingdetecting in the genome of the plant at least a first polymorphism. Themethod may, in certain embodiments, comprise detecting a plurality ofpolymorphisms in the genome of the plant. For example, a sample ofnucleic acid is obtained from a plant and a polymorphism or a pluralityof polymorphisms is detected in said nucleic acids. The method mayfurther comprise storing the results of the step of detecting theplurality of polymorphisms on a computer readable medium.

In one embodiment of the invention, the invention provides a method forproducing a seed of a variety derived from Purple Royale comprising thesteps of (a) crossing a carrot plant of variety Purple Royale with asecond carrot plant; and (b) allowing seed of a variety PurpleRoyale-derived carrot plant to form. This method can further comprisesteps of (c) crossing a plant grown from said variety PurpleRoyale-derived carrot seed with itself or a second carrot plant to yieldadditional variety Purple Royale-derived carrot seed; (d) growing saidadditional variety Purple Royale-derived carrot seed of step (c) toyield additional variety Purple Royale-derived carrot plants; andoptionally (e) repeating the crossing and growing steps of (c) and (d)to generate further variety Purple Royale-derived carrot plants, e.g.one or more times such as two times, three times, three or more timessuch as four times, five times, six times, seven times or even moretimes. For example, the second carrot plant is of an inbred carrotvariety, or alternatively, the second carrot plant in step c) is PurpleRoyale or the male or female parent plant of Purple Royale.

In still yet another aspect, the present invention provides a method ofproducing a plant or a seed derived from variety Purple Royale, themethod comprising the steps of: (a) preparing a progeny plant derivedfrom said variety by crossing a plant of variety Purple Royale with asecond plant; and (b) allowing seed of a variety Purple Royale-derivedcarrot plant to form. In one embodiment, the second plant is a plant ofan inbred line or of a wild accession of Daucus carota.

The method may additionally comprise: (c) crossing a plant grown fromsaid variety Purple Royale-derived carrot seed with itself or a secondcarrot plant to yield additional variety Purple Royale-derived carrotseed; (d) growing said additional variety Purple Royale-derived carrotseed of step (c) to yield additional variety Purple Royale-derivedcarrot plants; and optionally (e) repeating the crossing and growingsteps of (c) and (d) to generate further variety Purple Royale-derivedcarrot plants. For example, steps (c) and (d) may be repeated one ormore times such as one, two, three, four five or six, seven, eight,nine, ten, or more times to produce a further plant derived from theaforementioned starting variety. The further plant derived from varietyPurple Royale may be an inbred variety, and the aforementioned repeatedcrossing steps may be defined as comprising sufficient inbreeding toproduce the inbred variety. In the method, it may be desirable to selectparticular plants resulting from step (c) for continued crossingaccording to steps (b) and (c). By selecting plants having one or moredesirable traits, a plant is obtained which possesses some of thedesirable traits of the starting plant as well as potentially otherselected traits.

In certain embodiments, the present invention provides a method ofproducing a carrot root comprising: (a) obtaining a plant of theinvention, wherein the plant has been cultivated to maturity, and (b)collecting the carrot root from said plant.

The invention also provides for a food or feed product comprising orconsisting of a plant part described herein preferably a carrot root orpart thereof and/or an extract from a plant part described herein. Thefood or feed product may be fresh or processed, e.g., canned, steamed,boiled, fried, blanched and/or frozen, etc.

In still another embodiment the invention relates to a seed or plantproduced by selfing a plant of the invention.

In another aspect the invention refers to packages, e.g., a container, abag and the like, comprising at least one of the following: seeds orseed pellets of carrot variety designated Purple Royale, carrot plant(s)designated Purple Royale, parts thereof (e.g. roots), progeny of acarrot plant designated Purple Royale, parts thereof, EDV of a plantdesignated Purple Royale or parts thereof.

In one embodiment any of the plant of the invention comprises at least3, 4, 5 or more of the (average) morphological and/or physiologicalcharacteristics as described in Table 1 for the carrot variety PurpleRoyale.

Another aspect refers to a carrot plant, or a part thereof, having allor essentially all the physiological and/or morphologicalcharacteristics of a carrot plant of carrot variety Purple Royale whengrown under the same conditions.

Also provided are one or more progeny plants (offspring or descendants)of a carrot plant designated Purple Royale obtained by further breedingwith said variety designated Purple Royale. Said progeny plant(s)has/have essentially all physiological and/or morphologicalcharacteristics of variety Purple Royale when grown under the sameenvironmental conditions. In one embodiment, said progeny plant(s)has/have 3, 4, 5, 6, 7, 8, or more, or all of, the following (average)characteristics (see USDA descriptors) as described in Table 1.

Any embodiment discussed herein with respect to one aspect of theinvention applies to other aspects of the invention as well, unlessspecifically noted.

In the description and tables herein, a number of terms are used. Inorder to provide a clear and consistent understanding of thespecification and claims, the following definitions are provided:

The term “about” is used to indicate that a value includes the standarddeviation of error for the device or method being employed to determinethe value.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and to “and/or.”

When used in conjunction with the word “comprising” or other openlanguage in the claims, the words “a” and “an” denote “one or more”unless specifically noted.

The terms “comprise,” “have” and “include” are open-ended linking verbs.Any forms or tenses of one or more of these verbs, such as “comprises,”“comprising,” “has,” “having,” “includes” and “including,” are alsoopen-ended. For example, any method that “comprises,” “has” or“includes” one or more steps is not limited to possessing only those oneor more steps and also covers other unlisted steps. Similarly, any plantthat “comprises,” “has” or “includes” one or more traits is not limitedto possessing only those one or more traits and covers other unlistedtraits. The terms mentioned above also comprise the term “contain” whichis limited to specific embodiments. Thus, one embodiment of theinvention, when the terms “comprise,” “have” and “include” are used todescribe a plant, part thereof or a process, refers to an embodimentwherein the limiting term “contain” is used.

“Carrot” refers herein to a plant of the species Daucus carota and partsthereof, e.g., the (edible) root. The most commonly eaten part of acarrot is a root, although the greens are edible as well. A carrot is aroot vegetable plant, the root (carrot root) is usually orange in color,though purple, red, white, and yellow varieties exist, as well. At thetip of a carrot root is a thin taproot while at the other end (base) ofa carrot root the green is attached.

“Cultivated carrot” refers to plants of Daucus carota, i.e. varieties,breeding lines or cultivars of the species Daucus carota, cultivated byhumans and having good agronomic characteristics; preferably such plantsare not “wild plants”, i.e. plants which generally have much pooreryields and poorer agronomic characteristics than cultivated plants ande.g. grow naturally in wild populations. “Wild plants” include forexample ecotypes, PI (Plant Introduction) lines, landraces or wildaccessions or wild relatives of a species.

“USDA descriptors” are the plant variety descriptors described forcarrot in the “Objective description of Variety Carrot Daucus carota)”,ST-470-78 (as published by U.S. Department of Agriculture, AgriculturalMarketing Service, Science and Technology, Plant Variety ProtectionOffice, Beltsville, Md. 20705.

“UPOV descriptors” are the plant variety descriptors described forcarrot in the “Guidelines for the Conduct of Tests for Distinctness,Uniformity and Stability, TG/49/8 (Geneva 2007), as published by UPOV(International Union for the Protection of New Varieties and Plants) andis herein incorporated by reference in its entirety.

“Core” refers to the phloem and xylem of the root, i.e. the centralvascular tissue. “Cortex” refers to the non-vascular tissue surroundingthe core tissue.

“Genotype” refers to the genetic composition of a cell or organism.

“Phenotype” refers to the detectable characteristics of a cell ororganism, which characteristics are the manifestation of geneexpression.

As used herein, the term “plant” includes the whole plant or any partsor derivatives thereof, preferably having the same genetic makeup as theplant from which it is obtained, such as plant organs (e.g. harvested ornon-harvested carrot root), plant cells, plant protoplasts, plant celland/or tissue cultures from which whole plants can be regenerated, plantcalli, plant cell clumps, plant transplants, seedlings, hypocotyl,cotyledon, plant cells that are intact in plants, plant clones ormicropropagations, or parts of plants (e.g. harvested tissues ororgans), such as plant cuttings, vegetative propagations, embryos,pollen, ovules, flowers, leaves, seeds, clonally propagated plants,roots, taproots, stems, root tips, grafts, parts of any of these and thelike. Also, any developmental stage is included, such as seedlings,cuttings prior or after rooting, mature plants, roots or leaves.Alternatively, plant part may also include a plant seed which comprisesone or two sets of chromosomes derived from the parent plant.

“Harvested plant material” refers herein to plant parts (e.g. a rootdetached from the whole plant) which have been collected for furtherstorage and/or further use.

“Harvested seeds” refers to seeds harvested from a line or variety, e.g.produced after self-fertilization or cross-fertilization and collected.

A plant having “(essentially) all the physiological and/or morphologicalcharacteristics” means a plant having essentially all or all thephysiological and/or morphological characteristics when grown under thesame environmental conditions of the plant of Purple Royale from whichit was derived, e.g. the progenitor plant, the parent, the recurrentparent, the plant used for tissue- or cell culture, etc. The skilledperson will understand that a comparison between carrot varieties shouldoccur when said varieties are grown under the same environmentalconditions. For example, the plant may have all characteristicsmentioned in Table 1 when grown under the environmental conditionsoutlined herein with reference to the data of Table 1. In certainembodiments, the plant having “essentially all the physiological and/ormorphological characteristics” are plants having all the physiologicaland/or morphological characteristics of Table 1, except for certaincharacteristics, such as one, two or three, mentioned, e.g. thecharacteristic(s) derived from a converted or introduced gene or traitand/or except for the characteristics which differ in an EDV. So, theplant may have all characteristics mentioned in Table 1, except for one,two or three characteristics of Table 1, in which the plant may thusdiffer.

A plant having one or more or all “essential physiological and/ormorphological characteristics” or one or more “distinguishingcharacteristics” (such as one, two, three, four or five) refers to aplant having (or retaining) one or more, or all, or retaining all exceptone, two or three of the distinguishing characteristics mentioned inTable 1 when grown under the same environmental conditions thatdistinguish Purple Royale from most similar varieties Anthonina, PurpleElite, Purple Haze, and Deep Purple. For example, such distinguishingcharacteristics being selected from (but not limited to): 1) averageroot core thickness at midpoint cross-section, 2) average root diameterat midpoint, 3) carrot length (minus taproot), 4) cross-section interiorcolor of core and cortex, 5) haloing and zoning of the root at marketmaturity, 6) Petiole length from crown to first pinna, 7) average lengthof taproot, 8) blade color of leaf at harvest stage, 9) blade divisionsof leaf at harvest stage, 10) leaf petiole anthocyanin level and petiolepubescence at harvest stage, 11) average rood diameter at shoulder (seeUSDA descriptors, and 12) total anthocyanin levels.

The physiological and/or morphological characteristics mentioned aboveare commonly evaluated at significance levels of 1%, 5%, 8% or 10%significance level, when measured under the same environmentalconditions. For example, a progeny plant of Purple Royale may have oneor more (or all, or all except one, two or three) of the essentialphysiological and/or morphological characteristics of Purple Royalelisted in Table 1, or one or more or all (or all except one, two orthree) of the distinguishing characteristics of Purple Royale listed inTable 1 and above, as determined at the 1% or 5% significance level whengrown under the same environmental conditions.

Physiological and/or morphological characteristics which are“substantially equivalent” or “not significantly different” or “notsignificantly differ” refers to a characteristic that, when compared,does not show a statistically significant difference (e.g., p>0.05 usingANOVA) from the mean. Vice versa, “significantly different” or“statistically significantly different” refers to a characteristic that,when compared, does show a statistically significant difference (e.g.,p<0.05 using ANOVA) from the mean.

As used herein, the term “variety” or “cultivar” means a plant groupingwithin a single botanical taxon of the lowest known rank, whichgrouping, irrespective of whether the conditions for the grant of abreeder's right are fully met, can be defined by the expression of thecharacteristics resulting from a given genotype or combination ofgenotypes, distinguished from any other plant grouping by the expressionof at least one of the said characteristics and considered as a unitwith regard to its suitability for being propagated unchanged.

The terms “gene converted” or “conversion plant” in this context referto carrot plants which are often developed by backcrossing whereinessentially all of the desired morphological and/or physiologicalcharacteristics of parent are recovered in addition to the one or moregenes transferred into the parent via the backcrossing technique or viagenetic engineering. Likewise, a “Single Locus Converted (Conversion)Plant” refers to plants which are often developed by plant breedingtechniques comprising or consisting of backcrossing, wherein essentiallyall of the desired morphological and physiological characteristics of acarrot variety are recovered in addition to the characteristics of thesingle locus having been transferred into the variety via, e.g., thebackcrossing technique and/or by genetic transformation. Likewise, adouble-loci converted plant/a triple loci converted plant refers toplants having essentially all of the desired morphological and/orphysiological characteristics of given variety, expect that at two orthree loci, respectively, it contains the genetic material (e.g., anallele) from a different variety.

A variety is referred to as an “Essentially Derived Variety” (EDV) i.e.,shall be deemed to be essentially derived from another variety, “theinitial variety” when (i) it is predominantly derived from the initialvariety, or from a variety that is itself predominantly derived from theinitial variety, while retaining the expression of the essentialcharacteristics that result from the genotype or combination ofgenotypes of the initial variety; (ii) it is clearly distinguishablefrom the initial variety; and (iii) except for the differences whichresult from the act of derivation, it conforms to the initial variety inthe expression of the essential characteristics that result from thegenotype or combination of genotypes of the initial variety. Thus, anEDV may be obtained for example by the selection of a natural or inducedmutant, or of a somaclonal variant, or an off-type, or the selection ofa (natural) variant individual from plants of the initial variety,backcrossing, or transformation by genetic engineering. Such a variantmay be selected at any time, e.g. in the field or greenhouse, duringbreeding, during or after in vitro culture of cells or tissues, duringregeneration of plants, etc. The term EDV, thus, also encompassed a“phenotypic variant” derived from Purple Royale seed, plant tissue orcells. In one embodiment, an EDV is a gene converted plant.

“Plant line” is for example a breeding line which can be used to developone or more varieties.

“Progeny” (or “descendants”) as used herein refers to plants derivedfrom a plant designated Purple Royale. Progeny may be derived byregeneration of cell culture or tissue culture or parts of a plantdesignated Purple Royale or selfing of a plant designated Purple Royaleor by producing seeds of a plant designated Purple Royale. In furtherembodiments, progeny may also encompass plants derived from crossing ofat least one plant designated Purple Royale with another carrot plant ofthe same or another variety or (breeding) line, or with a wild carrotplant, backcrossing, inserting of a locus into a plant or selecting aplant comprising a mutation or selecting a variant. A progeny is, e.g.,a first generation progeny, i.e. the progeny is directly derived from,obtained from, obtainable from or derivable from the parent plant by,e.g., traditional breeding methods (selfing and/or crossing) orregeneration. However, the term “progeny” generally encompasses furthergenerations such as second, third, fourth, fifth, sixth, seventh or moregenerations, i.e., generations of plants which are derived from,obtained from, obtainable from or derivable from the former generationby, e.g., traditional breeding methods, regeneration or genetictransformation techniques. For example, a second generation progeny canbe produced from a first generation progeny by any of the methodsmentioned above. Especially progeny of Purple Royale which are EDVs orwhich retain all (or all except 1, 2 or 3) physiological and/ormorphological characteristics of Purple Royale listed in Table 1, orwhich retain all (or all except 1, 2, or 3) of the distinguishingcharacteristics of Purple Royale described elsewhere herein and in Table1, are encompassed herein.

The term “traditional breeding techniques” encompasses herein crossing,selfing, selection, double haploid production, embryo rescue, protoplastfusion, marker assisted selection, mutation breeding etc. as known tothe breeder (i.e. methods other than geneticmodification/transformation/transgenic methods), by which, for example,a genetically heritable trait can be transferred from one carrot line orvariety to another.

“Crossing” refers to the mating of two parent plants.

“Cross-pollination” refers to the fertilization by the union of twogametes from different plants.

“Backcrossing” is a traditional breeding technique used to introduce atrait into a plant line or variety. The plant containing the trait iscalled the donor plant and the plant into which the trait is transferredis called the recurrent parent. An initial cross is made between thedonor parent and the recurrent parent to produce progeny plants. Progenyplants which have the trait are then crossed to the recurrent parent.After several generations of backcrossing and/or selfing the recurrentparent comprises the trait of the donor. The plant generated in this waymay be referred to as a “single trait converted plant”.

“Selfing” refers to self-pollination of a plant, i.e., the transfer ofpollen from the anther to the stigma of the same plant.

“Regeneration” refers to the development of a plant from cell culture ortissue culture or vegetative propagation.

“Vegetative propagation”, “vegetative reproduction” or “clonalpropagation” are used interchangeably herein and mean the method oftaking part of a plant and allowing that plant part to form at leastroots where plant part is, e.g., defined as or derived from (e.g. bycutting of) leaf, pollen, embryo, cotyledon, hypocotyl, cells, nodes,protoplasts, meristematic cell, root, root tip, pistil, anther, flower,shoot tip, shoot, stem, petiole, etc. When a whole plant is regeneratedby vegetative propagation, it is also referred to as a vegetativepropagation.

The term “locus” (loci plural) means a specific place or places or asite on a chromosome where for example a gene or genetic marker isfound. “Linkage” or “genetic linkage” is the tendency of genes genes ormolecular markers that are located proximal to each other on achromosome chromosome to be inherited together during meiosis. Genes ormolecular markers whose loci are nearer to each other are less likely tobe separated onto different chromatids chromatids during chromosomalcrossover chromosomal crossover and are therefore said to be geneticallylinked.

“Marker” or “molecular marker” refers to a readily detectable DNAsequence or nucleotide, which may be genetically closely linked to agene or locus. Such closely linked markers can be used in MAS (markerassisted selection) of the gene or locus.

“Marker assisted selection” or “MAS” is a process of using the presenceof molecular markers, which are genetically linked to a particular locusor to a particular chromosome region, to select plants for the presenceof the specific locus or region.

The term “allele(s)” means any of one or more alternative forms of agene at a particular locus, all of which alleles relate to one trait orcharacteristic at a specific locus. In a diploid cell of an organism,alleles of a given gene are located at a specific location, or locus(loci plural) on a chromosome. One allele is present on each chromosomeof the pair of homologous chromosomes. A diploid plant species maycomprise a large number of different alleles at a particular locus.These may be identical alleles of the gene (homozygous) or two differentalleles (heterozygous).

As used herein, the terms “resistance” and “tolerance” are usedinterchangeably to describe plants that show no symptoms orsignificantly reduced symptoms to a specified biotic pest, pathogen,abiotic influence or environmental condition. These terms are also usedto describe plants showing some symptoms but that are still able toproduce marketable product with an acceptable yield.

“Tissue Culture” refers to a composition comprising isolated cells ortissues of the same or a different type or a collection of such cellsorganized into parts of a plant.

“Average” refers herein to the arithmetic mean.

The term “mean” refers to the arithmetic mean of several measurements.The skilled person understands that the appearance of a plant depends tosome extent on the growing conditions of said plant. Thus, the skilledperson will know typical growing conditions for carrot described herein.The mean, if not indicated otherwise within this application, refers tothe arithmetic mean of measurements on at least 10 different, randomlyselected plants or plant parts of a variety or line.

As used herein, the term “anthocyanin” refers to anthocyanidins withsugar group(s), mostly 3-glucosides of the anthocyanidins, though other3-glycosides and 5-glycosides are also known (Stintzing et al. AgricFood Chem. 2002. Vol 50. 6172-6181). The anthocyanins can be subdividedinto the sugar-free anthocyanidin aglycone and the anthocyaninglycosides. The difference in chemical structure that occurs in responseto changes in pH is the reason why anthocyanins are often used as pHindicators, as they change from red in acids to blue in bases (i.e.basic environment such as pH>7 or >8 or even pH>9 or >10 or >11).

The term “total anthocyanin level” or “total anthocyanin content” refersto the amount of anthocyanins in the root of the carrot at marketmaturity. Whenever reference is made to “total anthocyanin level”, thecombined level of all glycosides of anthocyanidin together is beingreferred to, without identifying individual anthocyanins. Differentparts of the root of a carrot may contain different levels ofanthocyanin e.g. sampling root shoulder vs root tips will give differentresults. The middle section (between top and shoulder) of a carrot givesa good sample of the anthocyanin level of the major part of a carrot.Total anthocyanin levels may also depend somewhat depending on the triallocation and sampling moment (e.g. stage of growth), however whenmeasured under the same conditions, the variation between genotypesremains relatively stable and the ranking of genotypes does not change.

The total anthocyanin level per carrot can be determined using methodsknown in the art for example by purifying fresh carrot (whole carrotroot, excluding tap root), optionally add an internal standard likemalvidin-3-galactoside chloride to determine extraction efficacy,extract with 10% formic acid in methanol five times, centrifuge theextracts and combine the supernatants. Anthocyanins can then bedetermined by HPLC e.g. by using a C18 column and a mobile phase A of10% formic acid in water and a mobile phase B of methanol using agradient of B from 0 to 55%. Anthocyanins can be identified by comparingretention times and UV or MS spectra with known anthocyanins. A standardresponse curve for quantifying the anthocyanin may be prepared fromcyanidin-3-glucoside.

The term “genetic elements conferring the total anthocyanin level”refers to all genes responsible for the total anthocyanin level in theroot of a carrot plant.

The term “genetic elements conferring the level of haloing and zoning”refers to all genes responsible for the level of haloing and zoning inthe root of a carrot plant.

Genetic elements conferring a specific trait may for example bedetermined using methods known in the art such as whole exome sequencing(also known as targeted exome capture) which is an approach thatinvolves using sequencing technology and sequence assembly tools topiece together all coding portions of the genome. The sequence is thencompared to a reference genome and any differences are noted. Afterfiltering out all known polymorphisms, synonymous changes, and intronicchanges (that do not affect splice sites), only potentially traitaffecting variants will be left. This technique can be combined withother techniques to further exclude potentially trait-affecting variantsshould more than one be identified. Alternatively, genetic approaches,such as gene mapping or Quantitative Trait Analysis (using e.g. BulkSegregant Analysis, as described by Michelmore et al. Proc Natl Acad SciUSA. 1991 Nov. 1; 88(21): 9828-9832 may be used to identify the numberand location of genes which determine the trait and molecular markerslinked to the genes or genome regions which determine the trait.

Seeds of the Carrot Variety Purple Royale.

Also provided are seeds of carrot variety Purple Royale, i.e. seeds fromwhich the variety can be grown.

In one embodiment, a plurality of Purple Royale seeds are packaged intosmall and/or large containers (e.g., bags, cartons, cans, etc.). Seedsmay be treated with one or more chemical compounds and/or biologicalcontrol agents (e.g. to improved germination, insecticidal-,acaricidal-, nematicidal- or fungicidal-compounds or compositions, etc.)and/or seeds may be primed. Biological control agents are one or moremicroorganisms which protect the seed or seedling against pathogens. Forexample, strains of bacteria and/or fungi, such as bacteria of thespecies of Streptomyces, Pseudomonas, Bacillus and Enterobacter or fungiof the species Phomopsis, Ectomycorrhizae, Trichoderma, Cladosporium andGliocladium.

Priming is a water-based process that is performed on seeds to increaseuniformity of germination and emergence from the soil, and thus enhancevegetable stand establishment. Methods how to prime carrot seeds arewell known in the art, see WO2008/107097, describing different primingmethods, such as hydro-priming (including drum-priming), osmo-primingand solid-matrix priming, which can be used. The priming process mayalso be combined with the chemical compounds or compositions and/orbiological control agent treatment, so seeds may e.g. be hydrated in afirst step, dried in a second step and treated in a third step with oneor more seed treatment compounds or compositions. Priming is alsosometimes referred to as seed conditioning.

Hydropriming includes those techniques in which seeds are allowed totake up water for a short period or at low temperatures, mostly at amplewater supply. These techniques are sometimes also referred to as soakingor steeping. With osmo-priming, the seeds are exposed to an osmoticsolution (see e.g. WO2008/107097).

With solid matrix priming (SMP), seeds are mixed with water and solidcarriers. Examples of solid carriers are vermiculite and diatomaceoussilica products. The water is taken up by the seeds as well as absorbedon the solid particle surfaces, which in this way control the wateruptake of the seeds. In addition to using particle-like carriers, SMPcan be carried out using, amongst others, moist towels, gunny bags,moist sand, sterilized compost or press mud as well.

So, in one aspect seeds of Purple Royale are provided wherein said seedsare primed seeds and/or chemically and/or biologically treated seeds,comprising one or more chemical compounds or compositions and/or one ormore biological control agents, selected from the group consisting of: acompound that improves germination, an insecticidal compound, anacaricidal compound, a nematicidal compound, and a fungicidal compound.

Breeding with Carrot Plants of the Invention

One aspect of the current invention concerns methods for crossing acarrot variety provided herein with itself or a second carrot plant andthe seeds and plants produced by such methods. These methods can be usedfor propagation of a variety provided herein or can be used to producecarrot seeds and the plants grown therefrom. Such seed can be producedby crossing two parent lines or varieties to produce the variety.

In one embodiment of the invention, the first step in “crossing”comprises planting seeds of a first and a second parent carrot plant,often in proximity so that pollination will occur for example, mediatedby insect vectors. Alternatively, pollen can be transferred manually.Where the plant is self-pollinated, pollination may occur without theneed for direct human intervention other than plant cultivation.

Also in accordance with the invention, novel varieties may be created bycrossing a plant of the invention followed by multiple generations ofbreeding according to such well known methods. New varieties may becreated by crossing with any second plant. In selecting such a secondplant to cross for the purpose of developing novel varieties, it may bedesired to choose those plants that either themselves exhibit one ormore selected desirable characteristics or that exhibit the desiredcharacteristic(s). Once initial crosses have been made, inbreeding andselection take place to produce new varieties. For development of auniform variety, often five or more generations of selfing and selectionare involved.

Uniform varieties of new varieties may also be developed by way ofdouble-haploids. This technique allows the creation of true breedingvarieties without the need for multiple generations of selfing andselection. In this manner, true breeding varieties can be produced in aslittle as one generation. Haploid cells, such as microspores, pollen,anther cultures, or ovary cultures can be used. The chromosomes of thehaploid cells may then be doubled autonomously, or by chemicaltreatments (e.g. colchicine treatment). From the double-haploid cells,fertile homozygous plants are obtained. In accordance with theinvention, any of such techniques may be used in connection with a plantof the invention and progeny thereof to achieve a homozygous variety.

Backcrossing can also be used to improve an inbred plant. Backcrossingtransfers one or more heritable traits from one inbred or non-inbredsource to an inbred that lacks those traits. The exact backcrossingprotocol will depend on the characteristic(s) or trait(s) being alteredto determine an appropriate testing protocol. When the term varietyPurple Royale is used in the context of the present invention, this alsoincludes plants modified to include at least a first desired heritabletrait such as one, two or three desired heritable trait(s).

This can be accomplished, for example, by first crossing a superiorinbred (recurrent parent) to a donor inbred (non-recurrent parent),which carries the appropriate genetic information (e.g., an allele) atthe locus or loci relevant to the trait in question. The progeny of thiscross is then mated back to the recurrent parent followed by selectionin the resultant progeny (first backcross generation, or BC₁) for thedesired trait to be transferred from the non-recurrent parent. Afterfive or more backcross generations with selection for the desired trait,the progeny is heterozygous at loci controlling the characteristic beingtransferred but are like the superior parent for most or almost allother loci. The last backcross generation would be selfed to give purebreeding progeny for the trait being transferred.

The parental carrot plant which contributes the desired characteristicor characteristics is termed the non-recurrent parent because it can beused one time in the backcross protocol and therefore need not recur.The parental carrot plant to which the locus or loci from thenon-recurrent parent are transferred is known as the recurrent parent asit is used for several rounds in the backcrossing protocol.

Many single locus traits have been identified that are not regularlyselected for in the development of a new inbred but that can be improvedby backcrossing techniques. Single locus traits may or may not betransgenic; examples of these traits include, but are not limited to,male sterility, herbicide resistance, resistance to bacterial, fungal,or viral disease, insect resistance, restoration of male fertility,modified fatty acid or carbohydrate metabolism, and enhanced nutritionalquality. These comprise genes generally inherited through the nucleus.

Direct selection or screening may be applied where the single locus(e.g. allele) acts in a dominant fashion. For example, when selectingfor a dominant allele providing resistance to a bacterial disease, theprogeny of the initial cross can be inoculated with bacteria prior tothe backcrossing. The inoculation then eliminates those plants which donot have the resistance, and only those plants which have the resistanceallele are used in the subsequent backcross. This process is thenrepeated for all additional backcross generations.

Although backcrossing methods are simplified when the characteristicbeing transferred is a dominant allele, recessive, co-dominant andquantitative alleles may also be transferred. In this instance, it maybe necessary to introduce a test of the progeny to determine if thedesired locus has been successfully transferred. In the case where thenon-recurrent variety was not homozygous, the F₁ progeny would not beequivalent. F₁ plants having the desired genotype at the locus ofinterest could be phenotypically selected if the corresponding trait wasphenotypically detectable in a heterozygous or hemizygous state. In thecase where a recessive allele is to be transferred and the correspondingtrait is not phenotypically detectable in the heterozygous of hemizygousstate, the resultant progeny can be selfed, or crossed back to the donorto create a segregating population for selection purposes.Non-phenotypic tests may also be employed. Selected progeny from thesegregating population can then be crossed to the recurrent parent tomake the first backcross generation (BC₁).

Molecular markers may also be used to aid in the identification of theplants containing both a desired trait and having recovered a highpercentage of the recurrent parent's genetic complement. Selection ofcarrot plants for breeding is not necessarily dependent on the phenotypeof a plant and instead can be based on genetic investigations. Forexample, one can utilize a suitable genetic marker which is closelygenetically linked to a trait of interest. One of these markers can beused to identify the presence or absence of a trait in the offspring ofa particular cross and can be used in selection of progeny for continuedbreeding. This technique is commonly referred to as marker assistedselection. Any other type of genetic marker or other assay that is ableto identify the relative presence or absence of a trait of interest in aplant can also be useful for breeding purposes. Procedures for markerassisted selection applicable to the breeding of carrot are well knownin the art. Such methods will be of particular utility in the case ofrecessive traits and variable phenotypes, or where conventional assaysmay be more expensive, time consuming or otherwise disadvantageous.Types of genetic markers which could be used in accordance with theinvention include, but are not necessarily limited to, Simple SequenceLength Polymorphisms (SSLPs), Simple Sequence Repeats (SSR), RandomlyAmplified Polymorphic DNAs (RAPDs), DNA Amplification Fingerprinting(DAF), Sequence Characterized Amplified Regions (SCARs), ArbitraryPrimed Polymerase Chain Reaction (AP-PCR), Amplified Fragment LengthPolymorphisms (AFLPs), and Single Nucleotide Polymorphisms (SNPs).

Carrot varieties can also be developed from more than two parents. Thetechnique, known as modified backcrossing, uses different recurrentparents during the backcrossing. Modified backcrossing may be used toreplace the original recurrent parent with a variety having certain moredesirable characteristics or multiple parents may be used to obtaindifferent desirable characteristics from each.

The variety of the present invention are particularly well suited forthe development of new varieties based on the elite nature of thegenetic background of the variety. In selecting a second plant to crosswith Purple Royale for the purpose of developing novel carrot varieties,it will typically be preferred to choose those plants that eitherthemselves exhibit one or more selected desirable characteristics orthat exhibit the desired characteristic(s). Examples of desirablecharacteristics may include, but are not limited to herbicide tolerance,pathogen resistance (e.g., insect resistance, nematode resistance,resistance to bacterial, fungal, and viral disease), cytoplasmic malesterility (CMS), improved harvest characteristics, enhanced nutritionalquality, increased antioxidant content, improved processingcharacteristics, high yield, improved characteristics related to thecarrot root flavor, texture, size, shape, durability, shelf life, andyield, increased soluble solids content, uniform ripening, delayed orearly ripening, seedling vigor, adaptability for soil conditions, andadaptability for climate conditions.

DEPOSIT INFORMATION

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity andunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the invention, as limited only bythe scope of the appended claims.

All references cited herein are hereby expressly incorporated herein byreference.

EXAMPLES Example 1 Development of Carrot Variety Purple Royale

The variety Purple Royale can be developed from two replications of 50plants each, from which 15 plants or plant parts can be randomlyselected and used to measure characteristics. Table 1 provides the USDAdescriptors for Purple Royale (this patent application), and ofAnthonina, Purple Elite, Purple Haze, and Deep Purple (commercialreference varieties).

In accordance with one aspect of the present invention, provided is aplant having essentially all physiological and/or morphologicalcharacteristics of a carrot variety Purple Royale. A description of thephysiological and/or morphological characteristics of carrot varietyPurple Royale is presented in Table 1.

A. Characteristics of Purple Royale

TABLE 1 The USDA descriptors for Purple Royale, Anthonina, Purple Elite,Purple Haze, and Deep Purple a field trial. The values are mean values.Purple Purple Purple Deep USDA Descriptor Royale Anthonina Elite HazePurple 1. TYPE: 6 6 6 6 6 1 = Amsterdam; 2 = Flakee; 3 = Berlicum; 4 =Chantenay; 5 = Danvers; 6 = Imperator; 7 = Nantes; 8 = Other (Specify)2. REGION OF ADAPTATION 7 7 7 7 7 IN THE UNITED STATES OF AMERICA: 1 =Northeast; 2 = Northwest; 3 = Southeast; 4 = Southwest; 5 = NorthCentral; 6 = South Central; 7 = Most Regions 3. MARKET MATURITY: NA NANA NA NA No. Days from Seeding to Harvest 4. PLANT TOP: (At Harvest NANA NA NA NA Stage) Habit: 2 2 2 2 2 1 = Erect; 2 = Semi-erect; 3 =Prostrate Plant Top Height (from 33.44 cm 40.69 cm 39.39 cm 39.46 cm40.15 cm Shoulder to Top of Crown) Plant Top Neck Diameter 1.23 cm 1.62cm 1.41 cm 1.55 cm 1.41 cm Top Attachment: 1 1 1 1 1 1 = Single; 2 =Multiple 5. LEAF: (At Harvest Stage) Name of Color Chart: RHS BladeColor: 1 = Light Green; 1 2 2 2 2 2 = Medium Green; 3 = Dark Green; 4 =Other (Specify) Color Chart Value Blue Green Green Green Green GreenColor Chart Value Number 120C N134B N134B 139B 135B Blade Divisions: 2 22 2 2 1 = Fine; 2 = Medium; 3 = Coarse Blade Length (Without 20.54 cm22.88 cm 23.53 cm 20.73 cm 20.20 cm Petiole) Petiole Length from Crown14.64 cm 22.75 cm 22.48 cm 21.69 cm 24.25 cm to First Pinna 5.50 cm 7.71cm 7.74 cm 7.73 cm 6.80 cm Petiole Anthocyanin: 2 2 2 2 2 1 = Absent; 2= Present Petiole Pubescence 2 1 (very 2 2 1 (very (heavy) slight)slight) 1 = Absent; 2 = Present 6. ROOT: (At Market Maturity) CortexThickness (Midpoint 0.50 cm 0.57 cm 0.72 cm 0.72 cm 0.56 cm X-Section)Core Thickness (Midpoint 1.23 cm 1.13 cm 1.16 cm 0.98 cm 1.12 cmX-Section) Carrot Length (Minus Taproot) 7.78 cm 9.76 cm 15.07 cm 10.64cm 9.42 cm Length of Taproot 11.51 cm 12.88 cm 6.66 cm 5.01 cm 8.35 cmDiameter at Shoulder 2.19 cm 2.23 cm 2.55 cm 2.43 cm 2.17 cm Diameter atMidpoint 0.59 cm 1.38 cm 1.80 cm 1.93 cm 1.52 cm Amount Exposed (Above 11 1 1 1 Ground): 1 = None; 2 = 1-10%; 3 = 11-20%; 4 = 21-30%; 5 =31-40%; 6 = >40% Shape: 2 2 2 2 2 1 = Round; 2 = Conic; 3 = CylindricalCollar: 2 2 2 2 2 1 = Sunken; 2 = Level; 3 = Square Shoulder: 1 1 1 1 11 = Rounded; 2 = Sloping; 3 = Square Base: 1 1 2 2 1 1 = Pointed; 2 =Medium; 3 = Blunt Surface Smoothness: 2 2 2 2 2 1 = Very Smooth; 2 =Dimpled or Corrugated Number of Secondary Root 2 2 2 2 2 Scars: 1 =None; 2 = Few; 3 = Many Appearance of Secondary Root 2 2 2 2 2 Scars: 1= Not Prominent; 2 = Prominent Halo: 2 3 3 3 3 1 = None; 2 = Faint; 3 =Prominent Zoning: 2 3 2 2 3 1 = None; 2 = Faint; 3 = Prominent FlavorHarshness: NA NA NA NA NA 1 = Very Harsh; Moderately Harsh; 3 = MildlyHarsh Flavor Sweetness: NA NA NA NA NA 1 = Not Sweet; 2 = ModeratelySweet; 3 = Very Sweet 7. COLORS: Color choices: Name of Color Chart: RHSBelow Ground Exterior Color N187A N187A N79A N79B N79A Shoulder N187AN187A N79A N79B N79A Skin N187A N187A N79A N79B N79A Cross-SectionInterior Color NA NA NA NA NA Core N79A 6D 5C N25C No color to describe** Cortex 79A 79A 79A N79A 79A 8. FLOWER: NA NA NA NA NA 9. DISEASEREACTIONS: NA NA NA NA NA 1 = Susceptible; 2 = Resistant; give races (ifknown) 10. INSECT REACTIONS: NA NA NA NA NA 1 = Susceptible; 2 =Resistant; give races (if known) 11. PHYSIOLOGICAL NA NA NA NA NAREACTIONS: 1 = Susceptible; 2 = Resistant Brix NA NA NA NA NA 1 =Susceptible and 2 = Resistant 12: TOTAL ANTHOCYANIN 26.26 ppm 4.01 ppm0.97 ppm 1.53 ppm 2.93 ppm LEVEL:

B. Brix *These are typical values. Values may vary due to environment.Other values that are substantially equivalent are also within the scopeof the disclosure. (--- equals not measured).

The degree Brix can be determined in Purple Royale and in the mostsimilar varieties Anthonina, Purple Elite, Purple Haze, and Deep Purple.

The method used was as follows:

C. Total Anthocyanin Level

The anthocyanin level of Purple Royale, Anthonina, Purple Elite, PurpleHaze, and Deep Purple was determined using the following protocols:

Anthocyanin Extraction

The skin will be peeled off the root slug and the root will be cut inhalf in a longitudinal direction. Half of the root will be diced intosmall pieces. About 1±0.0.1 g of the diced root will be weighed in anextraction bag and 5 mL of 0.025 M potassium chloride buffer (pH 1.0)will be added to the bag. Next, the root pieces will be grinded using apulverizer until chunks are not visible. 1 mL of the juice will bepipetted into a micro centrifuge tube. It will be centrifuged at10,000×g for 10 minutes at room temperature. The supernatant will beused for anthocyanin analysis.

Total Anthocyanin Measurement Using Spectrophotometer

Cyanidin-3-glucoside will be used as a standard to determine theextinction coefficient of total anthocyanin. The supernatant from purplecarrot extraction will be diluted in 0.025M potassium chloride buffer(pH 1.0) and absorbance will be read at 510 and 700 nm using UV-VISspectrophotometer with a sipper attachment. The anthocyaninconcentration will be calculated based on the absorbance level.

A separate trial will be performed to quantitatively determineanthocyanin levels in Purple Royale, Anthonina, Purple Elite, PurpleHaze, and Deep Purple using this protocol. Plants will be harvestedbefore bolting started. The recorded anthocyanin levels are the averageof about 30 individual roots.

Dry matter (DM) was determined by i) drying an empty aluminum pan in theoven for 1 hour and cool down in a desiccator cabinet; ii) weigh theempty aluminum pan (A=pan wt.); iii) weigh 1±0.0.1 g of diced root inthe pan (B=pan+fresh root wt.); iv) dry in the convection oven at 100°C. for 24 hours or in the vacuum oven at 90° C. for 18 hours; v) cool inthe desiccator cabinet; vi) weigh the pan with the dried root (C=pan+dryroot wt.); vii) calculate % dry matter ({(C−A)/(B−A)}*100%).

Example 2 Breeding History for Carrot Variety Purple Royale

Years 2014 and 2015—

Four different samples of Turkish origin dark purple carrots wereprovided by Sensient Colors in early 2014 for testing in Livingston,Calif. All subsequent years of production were also grown at the sametrial site with essentially the same cultural practices. Based uponprevious experience with carrots with an annual growth habit, plots(length=50′, bed width=40″) of these four lots were planted on 20 Sep.2014 in the Livingston Research Nursery. Annual carrots typically arebest suited for planting and growing during periods of shorteningdaylength to reduce premature bolting. When daylength begins to rapidlylengthen in the early spring, bolting frequently occurs rapidly androots convert fleshy tissue into a lignified taproot. Initialobservations of these plots noted extremely tall vegetative top growth,highly pubescent leaves, and multiple growing points at the crown. Plotswere lifted on 25 Feb. 2015 for steckling selections for potentialbreeding use. A wide array of root colors and shapes were observed withmany forked and split roots and high levels of lenticels. Root colorsranged from a light tan/yellow to violet to extremely dark purple.Intense selection pressure was placed upon selecting a straight,non-forked, smooth root with an Imperator type shape. Roots than metthese criteria were further selected for color intensity with the mostdesirable selections having an almost black coloration across bothphloem & xylem. Candidate roots were cut approximately 6″ beneath thecrown and visually sorted for color content at the tip. Elite roots(approximately 10%) were then laboratory screened for anthocyanincontent. Exactly 2 grams of tissue was grated and anthocyanin determinedusing a proprietary rapid extraction process. Approximately the highest20% (anthocyanin content) were selected for self-pollination net cagesand immediately replanted. The lot designated ‘A’ had the highest visuallevels of pigment and virtually all selections were made therefrom. Atotal of 28 cages were planted in a fashion where the primary king umbelwas enclosed in the cage to create a self-pollination and other umbelswere left on the outside to randomly pollinate with other purple/blackcarrots to create half-sib populations. At bloom, pupae of blue-bottleflies were inserted into these cages for pollination. Four larger cagescomposed of at least 50 roots/cage were also planted from roots withless extensive selection pressure. All seeds were harvested in mid-July2015.

Years 2015 and 2016—

On Sep. 30, 2015, 24 plots (10′×40″) were planted.

Twenty-two were from self-pollinations of elite roots and two were fromhalf-sib populations. Additionally, 0.2 acres were bulk planted from thevisually selected large cage 510 and two larger plots (50′×40″) withremnant seed of the original seed provided by Sensient Colors. Foliagefor plots derived from self-pollination cages was distinctly morecompact and consistent than the larger tops exhibited by the originalpopulation, the half-sibs, or the less aggressively selected largercages. Plots were lifted for evaluation and selection on Mar. 17, 2016.Plot number C60212 was identified as having the best combination of along, straight, smooth root combined with consistent dark pigment, whichwas further confirmed by laboratory analysis. Most other plots derivedfrom self- or mass-pollinations demonstrated inconsistent root shapesand color segregation ranging from tan/yellow roots to violet to darkpurple/black pigment. Altogether, eight individual cages for additionalself-pollination and one large cage (10′×20′) were replanted on 25 Mar.2016 for regrowth and seed production. Seed from all cages was harvestedin late July 2016.

Years 2016 and 2017—

Plots (50′×40″) of harvested 2016 seed were planted on 16 Sep. 16, 2016.Growing conditions that winter were extremely challenging. Near recordrainfall kept soil moisture at or above saturation and created diseasepressure and delayed growth. Stecklings were not lifted until 2 Apr.2017, which was the latest possible date due to the onset of bolting.Only limited selections were made. Relevant, however, was theanthocyanin test performed comparing the elite cage 668 vs. the originalunselected Turkish material. This advanced selection contained 51%higher anthocyanin content than the source population, better overallcolor uniformity, and more consistent root shape. Overall, four largecages (2 @ 20′×40′ and 2 @ 10×20′) were planted to increase seed of thiselite selection.

Years 2017 and 2018—

2 acres of bulk increase of seed from the four 2017 cages was planted onSep. 26, 2017 to produce stecklings for large scale open multiplication(0.6 acres) that was harvested in July 2018 with a yield ofapproximately 600 lbs. that was subsequently designated ‘Purple Royale’.Photos below provide reference to the visual appearance of selectedstecklings and the subsequent 2018 seed production. Steckling photosdemonstrate the color uniformity and intensity obtained by selectionprogram as well as improvements in root shape consistency. The currentbreeding generation is SM3 indicating one generation of self-pollinationfollowed by three generations of mass pollination.

What is claimed:
 1. A plant of carrot variety Purple Royale, whereinrepresentative seed of said carrot variety has been deposited under ATCCAccession No. ______.
 2. A plant part of the plant of claim 1, whereinthe plant part comprises at least one cell of said plant.
 3. A seed ofcarrot variety Purple Royale, wherein representative seed of said carrotvariety Purple Royale has been deposited under ATCC Accession No.______.
 4. A method of producing carrot seed, the method comprisingcrossing the plant of claim 1 with itself or a second carrot plant toproduce said carrot seed.
 5. The method of claim 4, the method furthercomprising crossing the plant of carrot variety Purple Royale with asecond, non-isogenic carrot plant to produce said carrot seed.
 6. An F₁carrot seed produced by the method of claim
 5. 7. A carrot plantproduced by growing the F₁ carrot seed of claim
 6. 8. A compositioncomprising the seed of claim 3 comprised in plant seed growth media. 9.The composition of claim 8, wherein the plant seed growth media is soilor a synthetic cultivation medium.
 10. A plant of carrot variety PurpleRoyale further comprising a single locus conversion, wherein said plantotherwise comprises all of the morphological and physiologicalcharacteristics of said carrot variety when grown under the sameenvironmental conditions, and wherein representative seed of said carrotvariety have been deposited under ATCC Accession No.
 11. A seed thatproduces the plant of claim
 10. 12. The seed of claim 11, wherein thesingle locus confers a trait selected from the group consisting ofincreased anthocyanin content, increased flower size, multiple petals,broad environmental adaptation, and insect and pest resistance, andresistance to bacterial, fungal, or viral disease.
 13. The method ofclaim 5, the method further comprising: a. crossing a plant grown fromsaid carrot seed with itself or a different carrot plant to produce seedof a progeny plant of a subsequent generation; b. growing a progenyplant of a subsequent generation from said seed of a progeny plant of asubsequent generation and crossing the progeny plant of a subsequentgeneration with itself or a second plant to produce seed of a progenyplant of a further subsequent generation; and c. repeating step (b) withsufficient inbreeding to produce seed of an inbred carrot plant that isderived from carrot variety Purple Royale.
 14. A method of producing acommodity plant product, the method comprising producing the commodityplant product from the plant of claim
 1. 15. The method of claim 15,wherein the commodity plant product is anthocyanin.